© Dr. Michael W. Pfaffl firstname.lastname@example.org
and summarize all technical aspects involved in quantitative
gene expression analysis using real-time PCR & RT-PCR.
( specificity, sensitivity, reproducibility, intra- & inter-assay variations,
kinetic PCR efficiency calculation, optimization strategy, etc.)
The GENE QUANTIFICATION web page illustrates the usefulness of a reliable quantification strategy, and the difference between absolute vs. relative quantification assays in kinetic PCR & kinetic RT-PCR.
RT-PCR is the technique of choice for analyzing
mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection
combines the ease and necessary exactness to be able to produce reliable
as well as rapid results.
The reverse transcription - polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low abundance mRNA, often obtained from limited tissue samples. However, real-time RT-PCR is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in RT and PCR. The recent introduction of fluorescence based kinetic (real-time) RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of the transcriptom.
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